A filamentous cytoskeleton in vertebrate smooth muscle fibers. (49/5414)

There are three classes of myofilaments in vertebrate smooth muscle fibers. The thin filaments correspond to actin and the thick filaments are identified with myosin. The third class of myofilaments (100 A diam) is distinguished from both the actin and the myosin on the basis of fine structure, solubility, and pattern of localization in the muscle fibers. Direct structural evidence is presented to show that the 100A filament constitute an integrated filamentous network with the dense bodies in the sarcoplasm, and that they are not connected to either the actin or myosin filaments. Examination of (a) isolated dense bodies, (b) series of consecutive sections through the dense bodies, and (c) redistributed dense bodies in stretched muscle fibers supports this conclusion. It follows that the 100-A filaments complexes constitute a structrally distinct filamentous network. Analysis of polyacrylamide gels after electrophoresis of cell fractions that are enriched with respect to the 100-A filaments shows the presence of a new muscle protein with a molecular weight of 55,000. This protein can form filamentous segments that closely resemble in structure the native, isolated 100-A filaments. The results indicate that the filamentous network has a structure and composition that distinguish it from the actin and myosin in vertebrate smooth muscle.  (+info)

Cardiac myosin from pig heart ventricle. Purification and enzymatic properties. (50/5414)

A method is described for the preparation of high purity myosin from the left ventricle of pig heart. The purified myosin was free from nucleic acid, actin, tropomyosin, troponin, the 150,000 molecular weight protein and other contaminants. Analyses of subunits in the purified myosin were carried out on 3.5% acrylamide gel with 0.1% SDS. Of the total protein present in myosin, 11.3% was in the light chains; light chain 1 (LC1), 5.9% and light chain 2 (LC2), 5.4%. Urea gel electrophoresis of the purified myosin showed three closely spaced bands corresponding to the 20,000 dalton, the charge-modified 20,000 dalton and the phosphorylated 20,000 dalton components. The properties of the Ca2+-activated and K+-activated ATPases [EC 3.6.1.3] of the purified myosin were also studied. The Km values were 27 and 55 muM and the Vmax values were 0.263 and 0.317 mumole P1/mg/min for the Ca2+-activated and K+-activated ATPases, respectively. The pH-activity profiles and the effects of SH modification were of the skeletal myosin type except that the activities were lower.  (+info)

Inability of the smallest light chain to bind to fetal fast muscle myosin. (51/5414)

1. The smallest light chain of myosin, g3, was not transferred from adult HMM to fetal myosin in alkali (pH 10.5) under conditions when the light chains dissociated from myosin. 2. The g3 isolated from adult myosin did not bind to fetal myosin at either pH 7.8 or 10.5.  (+info)

Trading force for speed: why superfast crossbridge kinetics leads to superlow forces. (52/5414)

Superfast muscles power high-frequency motions such as sound production and visual tracking. As a class, these muscles also generate low forces. Using the toadfish swimbladder muscle, the fastest known vertebrate muscle, we examined the crossbridge kinetic rates responsible for high contraction rates and how these might affect force generation. Swimbladder fibers have evolved a 10-fold faster crossbridge detachment rate than fast-twitch locomotory fibers, but surprisingly the crossbridge attachment rate has remained unchanged. These kinetics result in very few crossbridges being attached during contraction of superfast fibers (only approximately 1/6 of that in locomotory fibers) and thus low force. This imbalance between attachment and detachment rates is likely to be a general mechanism that imposes a tradeoff of force for speed in all superfast fibers.  (+info)

Insulin-like growth factor-1 attenuates the detrimental impact of nonocclusive coronary artery constriction on the heart. (53/5414)

Coronary artery narrowing (CAN) induces tissue injury, which may involve myocyte necrosis and apoptosis. Insulin-like growth factor (IGF)-1 may counteract cell death, modifying the detrimental effects of myocardial ischemia. On this basis, CAN was produced in female FVB.Igf+/- mice and nontransgenic littermates, and the animals were euthanized 7 days later. CAN consisted of an 82% reduction in the vessel luminal cross-sectional area in both groups of mice. Severe left ventricular dysfunction was present in CAN nontransgenic and transgenic mice, but heart and left ventricular weights increased more in littermates than in FVB.Igf+/- mice. Similarly, the changes in chamber volume and diastolic wall stress were greater in nontransgenic mice. Subacute tissue injury, represented by foci of replacement fibrosis, was 2.6-fold higher in CAN littermates than in FVB.Igf+/- mice. Ongoing myocyte necrosis was 5-fold greater in nontransgenic mice, whereas apoptosis was low and did not differ in the 2 groups of mice. In CAN nontransgenic mice, myocyte necrosis was 12-fold more frequent than apoptosis but, in CAN transgenic mice, these 2 types of cell death were comparable. alpha-Myosin and beta-myosin isoform mRNAs were affected by CAN, but alpha-myosin mRNA was reduced more in nontransgenic mice. In conclusion, myocyte necrosis and replacement fibrosis are the prevailing forms of myocardial damage induced by CAN. Constitutive overexpression of IGF-1 attenuates myocyte necrosis and tissue injury, having no effect on cell apoptosis. These factors limit ventricular dilation, myocardial loading, cardiac hypertrophy, and alterations in alpha- and beta-myosin isoform expression.  (+info)

Effect of beta-adrenoceptor activation on [Ca2+]i regulation in murine skeletal myotubes. (54/5414)

The present study used real-time confocal microscopy to examine the effects of the beta2-adrenoceptor agonist salbutamol on regulation of intracellular Ca2+ concentration ([Ca2+]i) in myotubes derived from neonatal mouse limb muscles. Immunocytochemical staining for ryanodine receptors and skeletal muscle myosin confirmed the presence of sarcomeres. The myotubes displayed both spontaneous and ACh-induced rapid (<2-ms rise time) [Ca2+]i transients. The [Ca2+]i transients were frequency modulated by both low and high concentrations of salbutamol. Exposure to alpha-bungarotoxin and tetrodotoxin inhibited ACh-induced [Ca2+]i transients and the response to low concentrations of salbutamol but not the response to higher concentrations. Preexposure to caffeine inhibited the subsequent [Ca2+]i response to lower concentrations of salbutamol and significantly blunted the response to higher concentrations. Preexposure to salbutamol diminished the [Ca2+]i response to caffeine. Inhibition of dihydropyridine-sensitive Ca2+ channels with nifedipine or PN-200-110 did not prevent [Ca2+]i elevations induced by higher concentrations of salbutamol. The effects of salbutamol were mimicked by the membrane-permeant analog dibutyryl adenosine 3', 5'-cyclic monophosphate. These data indicate that salbutamol effects in skeletal muscle predominantly involve enhanced sarcoplasmic reticulum Ca2+ release.  (+info)

Effect of Mg2+ on stress, myosin phosphorylation, and ATPase activity in detergent-skinned swine carotid media. (55/5414)

Smooth muscle contraction has a relatively high requirement for free magnesium (Mg2+). In this study we examined the effect of Mg2+ concentration ([Mg2+]) on Ca2+-dependent stress development and stress maintenance, myosin ATPase activity, and myosin light chain (MLC) phosphorylation levels in Triton X-100 detergent-skinned fibers of the swine carotid media. Increasing [Mg2+] in a stepwise fashion from 0.1 to 6 mM 1) decreased the magnitude and Ca2+ sensitivity of stress development but augmented the amount of stress maintained without proportional MLC phosphorylation, 2) produced a greater decrease in the Ca2+ sensitivity of MLC phosphorylation than that of stress development, and 3) decreased myosin ATPase activity. These findings demonstrate that Mg2+ differentially modulates the MLC phosphorylation-dependent development of stress and the MLC phosphorylation-independent maintenance of stress. We suggest that increases in [Mg2+] enhance stress maintenance by increasing [MgADP], thus increasing the number of cross bridges in a force-generating state, and by a direct effect on the pathway responsible for Ca2+-dependent, MLC phosphorylation-independent contractions.  (+info)

Atomic structure of scallop myosin subfragment S1 complexed with MgADP: a novel conformation of the myosin head. (56/5414)

The crystal structure of a proteolytic subfragment from scallop striated muscle myosin, complexed with MgADP, has been solved at 2.5 A resolution and reveals an unusual conformation of the myosin head. The converter and the lever arm are in very different positions from those in either the pre-power stroke or near-rigor state structures; moreover, in contrast to these structures, the SH1 helix is seen to be unwound. Here we compare the overall organization of the myosin head in these three states and show how the conformation of three flexible "joints" produces rearrangements of the four major subdomains in the myosin head with different bound nucleotides. We believe that this novel structure represents one of the prehydrolysis ("ATP") states of the contractile cycle in which the myosin heads stay detached from actin.  (+info)